In general chemistry, organic chemistry, and biochemistry, mixtures of radio labelled compounds may be separated by traditional liquid chromatography (LC), high performance liquid chromatography (HPLC), or paper chromatography. The constituents of the mixtures are thereby isolated into different fractions. The radioactivity of these fractions can be measured by mixing an aliquot with about two to four times its volume of scintillation liquid (generally referred to as a "scintillation cocktail") and thereafter counting light flashes produced by the radioactivity of the fractions. Thus liquid scintillation counting is known to be useful for measuring the concentration of a radio labelled species in a specimen or fraction.
Liquid chromatography (LC) is an established technique for separation and analysis of multi-component mixtures. A newer technique known as high performance liquid chromatography (HPLC) is preferred for the quantitation of thermally unstable and nonvolatile components. It has been made possible by the development of chemically bonded stationary phases. Solvent programming (i.e., gradient elution) is a common technique with HPLC. In normal bonded phase chromatography, the stationary phase is polar (as defined by the predominant functional group) and the mobile phase (elution solvent) is nonpolar. In this case, the nonpolar species (compounds in the mixture) which prefer the mobile phase exhibit lower retention and elute first. With reversed phase chromatography, the stationary phase is nonpolar and the mobile phase is polar. The solution elution order may be the reverse observed with normal bonded phase chromatography. An example of a solvent gradient for reverse phase chromatography is water mixed with increasing proportions of acetonitrile. The expected elution is in order of increasing hydrophobic character of the hydrocarbons in the specimen. If one or more of the components of the mixture being analyzed by HPLC is radio-tagged and to be quantified by liquid scintillation techniques, a consideration challenge is presented due to the continually changing composition of the solvent. The radioactivity of various fractions is determined by mixing an aliquot with about two to four times its volume of scintillation cocktail. Unfortunately, the counting efficiency may vary substantially as the composition of the solvent in the fraction varies.
Typically, liquid scintillation counting comprises the addition of a specimen or fraction that emits nuclear radiation to an organic liquid mixture which in turn emits light when intercepting the radiation. The flash of light is detected, for example by a photocell which is in a circuit that counts the flashes. The organic liquid comprises an aromatic solvent for capturing the energy of the radiation, primary and secondary fluors (fluorescent compounds) for converting the energy to a light flash and surfactants to enable the intimate admixture of the liquid with the specimen. See U.S. Pat. No. 4,124,527 for general background. An ideal scintillation cocktail would provide a very high degree of efficiency (number of flashes detected per number of radiated particles) over a range of specimen/cocktail mixtures and for a large number of specimen solvent types.
Prior liquid scintillation cocktails known to applicants for use in flow-through cells for measuring radioactivity in fractions from high performance liquid chromatography (HPLC) procedures, for example radio-tagged metabolites in pharmaceutical and biomedical research, have had the drawback of working with only certain solvents and then in only certain solvent/water mixtures. Some existing cocktails limit the selection of the HPLC solvents to methanol, isopropyl alcohol (IPA) or similar alcohols and sometimes tetrahydrofuran (THF). Other HPLC solvents such as tetrahydrofuran (THF) or acetonitrile form two phases with certain known cocktails and produce a nonuniform counting efficiency when blended with water in various proportions.